Project Summary B- and T-cell Non Hodgkin lymphomas Diffuse Large B cell lymphoma (DLBCL) is the most common lymphoma representing ~30% of all B cell Non Hodgkin lymphomas. DLBCL is classified into distinct molecular subtypes, including germinal center B cell-like (GCB) DLBCL, activated B cell-like (ABC) DLBCL, and primary mediastinal B cell lymphoma. ABC-DLBCL is the most chemo-resistant DLBCL subtype with a 5-year overall survival as low as 30% versus 59 and 64%, for GCB- DLBCL and primary mediastinal B cell lymphoma, respectively. Therefore, new treatments are needed to improve clinical outcome of the patients with DLBCL. The reasons for resistance in lymphomas are not well understood and could be attributed to, but not limited to, clonal heterogeneity, microenvironmental signaling, and are genetically complex lymphoproliferative diseases. massive activation of canonical and/or non-canonical pathways such as B cell receptor (BCR). Such complexity of drug response underscores the need for better understanding the role of complementary pathways, such as microenvironment signaling, in lymphomas. However, c urrent pre-clinical research in lymphoma has relied on testing compounds with suspension cultures of lymphoma cell lines in tissue culture plates, without taking into account the lymphoid microenvironment, where these cancers arise and reside. The overall goal of this IMAT R33 proposal is advanced development and validation of organoids platform technology for lymphoma cell lines and patient-derived xenografts, and answer unsolved questions related to DLBCL resistance and heterogeneity. This propose research will develop and validate an experimental therapeutics platform, named LETSSGo (Lymphoma-on-chip Engineered Technology for Single-Organoid Sequencing and Genomics). LETSSGO is an technology to culture lymphoma cell lines and patient derived xenografts in a microscale lymphoid-like microenvironment and integrate with single organoids genomic analysis by means of DNA barcoding. Following 3 aims will be pursued: Aim 1: Engineer and validate a lymphoma-specific mico-organoids platform for single and multi-cell encapsulation of ABC-DLBCL and GCB-DLBCL cell lines; Aim 2: Integrate drug treated micro- organoids with DNA barcoding for single-organoid genomics to determine tumor resistance as a function of tumor size and cell number; Aim 3: Engineer lymphoma patient derived xenograft micro-organoids of DLBCL and determine growth rate and heterogeneity in gene expression and DNA methylation as compared to mouse PDXs If successful, our technology will change the way scientists understood initiation and progression of lymphomas, enable mechanistic understanding of the role of tumor microenvironment and clonal heterogeneity, provide prognostic values and increase ?predictive power? of pre-clinical studies for drugs in development, and importantly, will allow a faster and more rational screening and translation of therapeutic regimens.